Fluorescence resonance energy transfer (FRET) quenching is a phenomenon in which an energy donor, a reporter excited by light, transfers its energy to an energy acceptor that is a quencher neighboring thereto (J. R. Lakowicz, Principles of fluorescence spectroscopy, 1999, Kluwer Academic/Plenum Publishers, New York). Herein, the efficiency of energy transfer has a close relation with the distance between the reporter and the quencher, is inversely proportional to the sixth power of the distance between the reporter and the quencher. Thus, if the distance between the two is longer than a certain distance, fluorescence resonance energy transfer (FRET) quenching will not occur.
In an example of fluorescence resonance energy transfer quenching, if a TAMRA fluorophore is used as the energy acceptor (quencher), light energy from the energy donor is absorbed into the absorption region of the energy acceptor. The absorbed energy becomes an excited state and is emitted in the emission region of the energy acceptor.
The energy transfer process in this quenching phenomenon occurs even when the quencher is composed of an azo compound. However, light emission from the quencher that is the energy acceptor does not occur. A quencher such as an azo compound in this transfer process differs from the above TAMRA fluorophore in that it emits energy in place of light by the action of heat or other agent. This quencher such as an azo compound is called “dark quencher”. The dark quencher emits heat energy in place of light energy during energy transfer, and thus a dye having no fluorescence. A fluorescent quencher can increase background noise, because the fluorescence spectra of the quencher and the reporter can overlap. However, the dark quencher provides a solution to this disadvantage, because it has no fluorescence, and thus causes no noise. In addition, the dark quencher provides an advantage in that two or more reporter-quencher probes can be used.
Representative compounds having a dark quencher structure BHQ1, BHQ2 and BHQ3, which are BHQ quencher derivatives (WO2001/086001A1). These quenchers comprise a diazo bond between dialkylaniline acting as an electron donor and an aromatic compound acting as an electron acceptor, and absorb fluorescence in a wavelength region of about 500-700 nm. However, a quencher having a salt structure is unstable during oligomer synthesis and purification, and an example thereof is BHQ3.
Other examples may include BBQ quencher (WO2006/084067A2) and quinone-based Iowa black quencher (U.S. Pat. No. 7,504,495B2). However, these quenchers have poor wavelength absorption ability compared to the above-described BHQ quenchers.
Meanwhile, quenchers are linked with fluorophores and used mainly in the chemical or biological field. In order to detect a target molecule, protein or nucleic acid, a molecule capable of binding complementarily thereto, a quencher and a fluorophore may be linked with one another, and the emission of fluorescence therefrom may be measured to determine whether the target material is present. This plays a great role in advanced development in the chemical and biological fields. A representative example of the application of the fluorescence resonance energy transfer theory in the biological field is the use of probes in real-time polymerase chain reaction (RT-PCR). The probes are used to detect specific regions of nucleotide sequences, and have recently been frequently used in real-time polymerase chain reaction (RT-PCR).
Examples of the probes include a TaqMan probe, a molecular beacon probe and the like. The TaqMan probe is a probe comprising a reporter (energy donor) attached to one end and a quencher attached to the other end. During polymerase chain reaction, the reporter escapes the influence of the quencher and emits its characteristic fluorescence. The level of fluorescence increases in proportion to the progression of polymerase chain reaction. The level of fluorescence is quantified and used as an indirect measure. The molecular beacon probe is hairpin-shaped and composed of a stem and a loop. When the molecular beacon probe is attached to a specific region during polymerase chain reaction, the structure is disassembled, and the reporter escapes the influence of the quencher, and emits its fluorescence.
The quencher can explain energy transfer from the reporter in the probe based on the quenching phenomenon of the fluorescence resonance energy transfer theory. Thus, the relationship between the reporter and the quencher in the probe plays an important role in the performance of the probe. If the quencher absorbs fluorescence in a broad wavelength region from the reporter linked to the probe, the limitation of fluorophores that can be used will be eliminated, and an excellent probe that can easily achieves detection can be fabricated. In other words, if the quencher absorbs fluorescence in a broader wavelength range, it may be linked with various fluorescent dyes to prepare probes.
Accordingly, the present inventors have made extensive efforts to develop a quencher having a higher efficiency, and as a result, have found that a quencher comprising a novel azo compound has excellent quenching properties, thereby completing the present invention.